The well-documented increase on pollutants from a wide variety of sources presents a potential threat to all living organisms. As the quantity and diversity of potentially toxic and/or carcinogenic chemicals entering the environment increases so does the need to develop new monitoring and toxic evaluation techniques that are cost effective, reliable and rapid. In addition, new techniques that reduce or replace whole animal/mammalian studies in early stages of product testing need to be developed, validated, and accepted into the regulatory process. Primary cultures of mammalian hepatocytes have proved invaluable for studying mechanisms of pharmacotoxicity and exhibit several advantages over freshly isolate cells or established cell lines: 1) the cell ability to recover from isolation stress and resume a function similar to that seen in vivo, and 2) the retention of xenobiotic metabolizing ability. For toxicological or carcinogenicity testing applications and use in aquatic monitoring programs in vitro system using juvenile cells will be important. Usually juveniles are more sensitive to the presence toxicants that adults, hence the use of juvenile hepatocytes may provide a more sensitive initial screening system than could be obtained with adult cells. Although the juvenile catfish cellular system appears to be a good in vitro model for aquatic toxicity testing, its sensitivity is limited as catfish exhibit high xenobiotic tolerance limits in vivo. Thus, the overall objective of this proposed project centers around: 1) developing a juvenile trout primary hepatocyte culture that can be maintained in a chemically defined serum-free medium, and 2) determining whether these cells could be used to reduce or replace mammalian hepatocytes in early stages of safety testing. It is proposed to use ATP levels, mitochondrial dehydrogenase activity, and lactate dehydrogenase leakage as indicators of cytotoxicity and metabolic competency, and trypan blue dye exclusion and neutral red dye uptake as measures of viability and cell survival. Specifically of interest is determining whether juvenile trout cells can serve as a screening tool for compounds that require metabolic activation: a) acetaminophen, b) aflatoxin, c) benzopyrene, and d) cyclophosphamide. Specific questions include: 1) Are ATP levels correlated with cell survival? 2) Can the MTT dye reduction assay, which indirectly measures mitochondrial dehydrogenase activity, be used to accurately assess mitochondrial functioning; 3) Do MTT levels correlate with measured ATP levels and other measures of cell viability?; 4) Can a quantitative, automated LDH microassay system, that would utilize a microplate reader to yield a cost and time effective method of directly assessing cytotoxicity due to a loss of membrane integrity, be developed; and 5) Can these proposed parameters accurately quantify the cytotoxic effects of compounds that must undergo metabolic activation? An important secondary purpose of this proposal is associated with the enhancement of undergraduate biomedical research at Canisius College, and the ability to involve and train undergraduate students in the principles of approaching and solving important ecotoxicological problems in the biomedical sciences. The long range goals of this project include examination of: 1) mechanisms of detoxification (role of glutathione, induction of mixed function oxidases, etc.), and 2) the suitability of applying this in vitro model to monitoring programs for aquatic systems.